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. 2012 Aug 28;109(35):13978-83.
doi: 10.1073/pnas.1201882109. Epub 2012 Aug 13.

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Sang-Hee Shim  1 , Chenglong Xia, Guisheng Zhong, Hazen P Babcock, Joshua C Vaughan, Bo Huang, Xun Wang, Cheng Xu, Guo-Qiang Bi, Xiaowei Zhuang
Affiliations

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Sang-Hee Shim et al. Proc Natl Acad Sci U S A. .

Abstract

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Photoswitching behavior of small-molecule probes for four membrane structures in live cells. BS-C-1 cells were labeled with (A) DiI for the plasma membrane, (B) MitoTracker Red for mitochondria, (C) ER-Tracker Red for the ER, (D) LysoTracker Red for lysosomes. Top row: Images taken with weak 561-nm illumination to excite fluorescence from these probes without switching them off appreciably. Middle row: Images taken after strong 561-nm illumination (approximately 10 kW/cm2), which turned the probes off efficiently. Bottom row: Images taken with weak 561-nm light after 405-nm light was used to turn the probes on again. Image contrasts in the middle and bottom rows were either the same as the images in the top row or increased by 6 times, as indicated. The incomplete recovery of ER- and LysoTrackers immediately after switching-off did not substantially degrade the STORM images due to the high labeling density. The recovery of ER- and LysoTrackers slowly increased and reached about 100% after 15 min of 405-nm illumination. Scale bars, 1 μm.
Fig. 2.
Fig. 2.
STORM images of four membrane organelles/structures in live cells. Conventional (i) and STORM (ii) images of (A) the plasma membrane labeled with DiI in a hippocampal neuron, (B) mitochondria labeled with MitoTracker Red in a BS-C-1 cell, (C) the ER labeled with ER-Tracker Red in a BS-C-1 cell, and (D) lysosomes labeled with LysoTracker Red in a BS-C-1 cell. The conventional fluorescence images were taken immediately before STORM imaging with a low excitation intensity to avoid switching off the dyes appreciably. The STORM images were acquired in 15 sec (A), 10 sec (B and C) and 1 sec (D). Arrowheads in A indicate spine necks along with their measured widths. Scale bars, 1 μm.
Fig. 3.
Fig. 3.
Plasma membrane dynamics in dendrites of a live hippocampal neuron at 37 °C. (A) Morphological changes of dendritic structures captured by a series of 10-sec STORM snapshots. Green arrowhead: A growing spine or filopodium. Blue arrowhead: An extending filopodium. Purple arrowhead: A retracting filopodium. (B–E) High-density single-particle-tracking of DiI. (B) Molecular trajectories lasting at least 15 camera frames (2 ms per frame) colored by their diffusion coefficients, D, according to the color map on the Right. (C) Diffusion coefficients in different dendritic structures. Error bars indicate standard errors: N = 613 traces for shaft; N = 90 traces for spine/filopodia. (D) A zoom-in of the boxed region in B. (E) Local distribution of diffusion coefficients within the filopodium in the dashed box in D. Error bars indicate standard errors (N = 7–18). Scale bars, 1 μm.
Fig. 4.
Fig. 4.
Mitochondrial dynamics in a live BS-C-1 cell. (A) A 6-sec STORM snapshot shows thin tubes connecting neighboring mitochondria (indicated by yellow arrowheads). (B) Width distribution of inter-mitochondria tubules taken from multiple cells. Black bars: All tubules. Green bars: Tubules prior to fission. Red bars: Tubules after fusion. (C) Fission (green arrowheads) and fusion (red arrowheads) events captured by a time-series of 2-sec STORM snapshots (i) and corresponding conventional images (ii). While the images were acquired continuously (Movie S1), only every other 2-sec snapshot is displayed here to save space. Scale bars, 500 nm.
Fig. 5.
Fig. 5.
ER dynamics in live BS-C-1 cells. (A) A time-series of 10-sec STORM snapshots. Blue arrowheads: Tips of extending tubules. (B) A composite image containing all snapshots in A with each localization colored by its time of appearance according to the color map on the Right. (C) Distribution of the widths of ER tubules. Green bars: Newly extended tubules. Red bars: Old tubules that had already existed for at least 2 min. (D) A time-series of 2-sec STORM snapshots. Blue arrowheads: Extending tubules. Purple arrowheads: Retracting tubules. Yellow arrowheads: Extending sheets. (E) A composite image containing all snapshots in D with each localization colored by the time of its appearance. Scale bars, 500 nm.
Fig. 6.
Fig. 6.
Two-color STORM images of mitochondria (green) and the ER (magenta) in a live BS-C-1 cell. The snapshots are 10 sec long. The ER tubules at the mitochondrial fission site are indicated by green arrowheads. Scale bars, 500 nm.
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References

    1. McMahon HT, Gallop JL. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature. 2005;438:590–596. - PubMed
    1. Shibata Y, Hu J, Kozlov MM, Rapoport TA. Mechanisms shaping the membranes of cellular organelles. Annu Rev Cell Dev Biol. 2009;25:329–354. - PubMed
    1. Detmer SA, Chan DC. Functions and dysfunctions of mitochondrial dynamics. Nat Rev Mol Cell Biol. 2007;8:870–879. - PubMed
    1. Hell SW. Microscopy and its focal switch. Nat Methods. 2009;6:24–32. - PubMed
    1. Heintzmann R, Gustafsson MGL. Subdiffraction resolution in continuous samples. Nat Photonics. 2009;3:362–364.

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